Antioxidant and Enzyme Inhibitory Potential of Streptomyces sp. G-18 Grown in Various Media

Streptomyces are bacteria well known for producing bioactive secondary metabolites which are commonly found in diverse habitats. The biosynthesis of metabolites from Streptomyces is influenced by various factors such as the growth medium, environmental conditions, and gene regulation. This study aimed to investigate the influence of different growth media on biomass production and the antioxidant and enzyme inhibitory potential of a crude extract obtained from Streptomyces sp. G-18 isolated from high altitudinal soil of Nepal. The highest dry weight growth was observed in R2YE medium (184 mg/L), followed by R5 (144 mg/L), YEME (38 mg/L), and R5M media (30 mg/L). The crude extract showed notable antioxidant activities against free radicals. The highest alpha-amylase inhibition was observed in the R2YE medium, and worthy lipase and tyrosinase inhibition was observed in the YEME medium. However, only the R2YE medium exhibited inhibitory potential against elastase and acetylcholinesterase, while crude extracts from R5, YEME, and R5 modified did not show any such activity. Overall, our findings suggest that the production of bioactive secondary metabolites in Streptomyces sp. G-18 was significantly influenced by the growth medium. This strain may be a promising source of enzyme inhibitors with potential applications in the pharmaceutical and cosmetic industries.


Introduction
Streptomyces, a Gram-positive bacterial genus in the family Streptomycetaceae and order Actinomycetales, has displayed a wide range of biochemical properties [1]. Streptomyces is distinguished from other bacteria in that it does not develop as a typical bacterial bacillary coccoid form but rather in a flamentous or mycelial form. Actinomycetes are unique in that they can grow on a wide variety of substrates present in the soil, including plant polymers such as chitin, cellulose, and hemicellulose, as well as animal products such as difcult-to-degrade insects [2]. Tey are unusual in the bacterial community due to their ability to survive in extreme environmental conditions of high pH, high temperature, and water stress [3]. Tey can be found in practically any environment, from the deep sea to the high mountains [4]. Streptomyces' most fascinating characteristic is its capacity to synthesize bioactive secondary metabolites that have a wide range of biochemical properties such as antifungals, antivirals, antitumoral, antihypertensives, immunosuppressants, and antibiotics [5]. Streptomyces are thought to be responsible for roughly one-third of the thousands of naturally occurring antibiotics [6]. Many secondary metabolites, including antibiotics, are produced in tandem with morphological distinction [7]. Streptomyces' secondary metabolites hinder a variety of biological processes; Streptomyces strains outperform other actinomycetes strains in terms of their capacity to generate a high number and variety of bioactive secondary metabolites. Tese secondary metabolites likely allow Streptomyces species to compete with other microbes, even those of the same genera [8].
Streptomyces has historically played a signifcant role in the identifcation of signifcant bioactive secondary metabolites, such as antibiotics, immunosuppressive medications, anticancer medicines, and other physiologically active chemicals [9]. Unfortunately, throughout the past few decades, comparable and well-known chemicals from terrestrial Streptomyces have continued to be discovered. In order to fnd novel bioactive compounds, it is advantageous to look for promising microorganisms in unknown or underexploited natural habitats with diferent isolation processes [10]. Given this, a lot of efort has been paid in recent years to the isolation of possible Streptomyces species in habitats with more severe settings, such as deep sea, desert, arctic, and volcanic environments. Several metabolites from Streptomyces are found to be efective in the formulation of antibiotics and several other medicines. Among the other uses, these metabolites are found efective in biochemical activities. Several Streptomyces have shown enzyme production activities (amylase, lipase, catalase, cellulose, protease, and asparaginase) that are of industrial importance [11]. Tese enzymes are being used in pharmaceuticals, laundry, cosmetics, paper, textile, fermentation processes, and wastewater treatment industries [12,13]. In addition to this, several metabolites from Streptomyces are investigated for their potential antioxidant, antiinfammatory, anticancer, antimicrobial, and several enzyme inhibitory potentials as well [14].
Inhibition of important enzymes is an efective strategy for the treatment and management of various diseases including Alzheimer's disease (AD) [15], diabetes mellitus (DM) [16], and dermatological disorders [17] in humans. Cholinesterase inhibitors, for example, raise acetylcholine levels in the brain, enhancing cognitive function in Alzheimer's patients [18]. Inhibitors of carbohydrate-digesting enzymes (alpha-amylase and alpha-glucosidase) help manage blood glucose levels in diabetic patients [19]. Similarly, inhibition of elastase and tyrosinase, which are involved in skin aging and pigmentation, are critical for cosmetic improvement [20,21]. As a result, numerous synthetic inhibitors are utilized in clinical trials, but their efcacy is limited and they have some negative efects [22,23]. Continuing eforts are being made to screen and create novel inhibitors from natural sources that are both efective and have fewer negative efects. In this study, we evaluated the antioxidant and enzymes inhibition potential of extract from Streptomyces sp. (G-18) grown and extracted on four diferent growth media.

Soil Collection and Species Isolation.
To isolate Streptomyces species, soil samples were obtained from Gosaikunda, Langtang National Park, Rasuwa, Nepal (28.0820°N, 85.4150°E). Streptomyces species were isolated using the serial dilution technique in an ISP2 medium using a sterile spreader as described previously [13].

Extract Preparation and Growth Analysis. Te growth of
Streptomyces sp. (G-18) in diferent media was evaluated in 24 hrs intervals for a total 168 hrs. In brief, from each of the media, 1 mL of extracts was taken and dried to determine dry weight of the bacterial growth and repeated for seven days. Growth curve analyses were carried out by plotting the bacterial growth curve [25]. Furthermore, after 168 hrs of growth, metabolites were extracted using ethyl acetate as a solvent, dried using a vacuum evaporator, and used for antioxidant and enzyme inhibition assays.

Antioxidant
Assays. Antioxidant activity of extracted metabolites was determined by following standard for 2, 2diphenyl-1-picrylhydrazyl (DPPH) and 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assays [19]. To measure the DPPH free radical scavenging, bacterial extracts at various concentrations (10,20,40,60,80, and 100 μg/mL) were mixed with a 0.1 mM DPPH solution in a ratio of 1 : 3 and incubated for 30 min at dark. Te absorbance was measured using a UV-visible spectrophotometer at 517 nm (Shimadzu UV-1800). For the ABTS assay, a sample of 1 mL of bacterial extracts at various concentrations (10,20,40,60,80, and 100 μg/mL) was combined with 3 mL of ABTS working solution (mixture of ABTS and sodium potassium tartrate) and allowed to incubate in darkness for 10 min. Te decrease in absorbance at the wavelength of 720 nm was measured using a UV-visible spectrophotometer. Ascorbic acid was used as a reference for both DPPH and ABTS. Moreover, methanolic DPPH was used as a control for the DPPH assay, and methanolic ABTS was used as a control for the ABTS assay.
2.6. Alpha-Amylase Assay. Te 3,5-dinitrosalicylic acid (DNSA) method was used to determine α-amylase inhibition activity [19] in which 200 μL each of extracts (20-360 μg/mL) and 3 unit/mL α-amylase (Sigma-Aldrich, USA) were mixed and incubated for 15 min at 37°C. An additional 5 min of incubation at 37°C was carried out after the addition of 200 μL of 1% starch solution. Ten, a 200 μL of DNSA solution was added to it and the whole reaction mixture was heated for 10 min at 95°C. Te fnal volume was adjusted to 5 mL with the addition of distilled water. Te absorbance was measured at λ � 540 nm in a UV-visible spectrophotometer. Te results were compared with the activities of acarbose and 1% dimethyl sulfoxide (DMSO) was used as a positive control.

Lipase Inhibition Assay.
Te standard method of measuring the lipase activity was adapted by using 4-nitrophenyl butyrate (p-NPB) as a substrate [26]. In brief, 80 μL of extracts (10-160 μg/mL) were mixed with 40 μL of substrate solution (10 mM of p-NPB in ethanol) and 80 μL of the lipase enzyme (2.5 mg/mL in 0.1 M phosphate bufer, pH 8.0). After incubation (20 min; 37°C), absorbance was observed at 405 nm using a 96-microplate reader (BioTek, EPOCH). Te results were compared with the activities of orlistat and 1% DMSO was used as a positive control.

Tyrosinase Inhibition
Assay. Te l-3,4-dihydroxyphenylalanine (L-DOPA) substrate was used to determine tyrosinase inhibition activity [27]. In brief, 40 μL extracts (10-160 μg/mL) in potassium phosphate bufer (0.05 M, pH 6.5) were mixed with 15 μL of mushroom tyrosinase enzyme (500 U/mL) and incubated at 27°C for 10 min. Ten, by adding 20 μL of L-DOPA (5 mM), the reaction volume was maintained to 200 μL by the addition of potassium phosphate bufer and incubated for an additional 30 min. Absorbance was measured at 492 nm in a 96microplate reader. Te results were compared with the activities of kojic acid.
2.9. Elastase Inhibition Assay. Te N-succinyl-Ala-Ala-pnitroanilide (AAAPVN) substrate was used to assess the elastase inhibitory activity, following the Ellman method with minor modifcations [28]. In brief, extracts of (10-160 μg/mL) were mixed with porcine pancreatic elastase (0.05 U/mL) and 50 g/mL AAAPVN and incubated for 30 min at 25°C. With the addition of 0.2 M Tris-HCl bufer (pH 8.0), the fnal reaction volume was maintained at 200 μL. In a 96-microplate reader, absorbance was measured at 410 nm. As a positive control, 1% DMSO was used, and the results were compared with the activities of quercetin.

Cholinesterase Inhibition
Assay. Te analysis of inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was conducted [29]. To begin, 0.05 U/mL AChE or 0.5 U/mL BChE was combined with extracts (10-160 μg/mL) and incubated at 25°C for 15 min. Te reaction mixture was then supplemented with 1 mM acetylthiocholine iodide or 1.5 mM butyrylcholine iodide and 0.5 mM of 5,5′-dithiobis-(2-nitrobenzoic acid) (DNTB). By adding 0.1 M sodium phosphate bufer (pH 8.0), the total reaction volume was maintained at 200 μL. Te absorbance was determined at 412 nm using a 96-microplate reader. 1% DMSO was used as positive control and results were compared with the activities of galantamine.

Statistical Analysis.
Te experiments were performed in triplicate for each analysis and the results are presented as mean ± standard deviation (mean ± SD). Te percentage of radicals scavenging and enzymes inhibition was determined by calculating the diference between the values of positive control and examined extracts as shown in the following formula [29]. Moreover, the inhibitory concentration at which absorbance is 50% (IC 50 ) values were calculated by linear regression analysis of the percentage of radicals scavenging and percentage of enzymes inhibition. Te signifcant diference in the mean value of examined extracts was analyzed by the one-way analysis of variance (one-way ANOVA), post hoc Tukey, and multiple comparison test at 95% confdence level (p < 0.05) by using SPSS version 26.

% Inhibition �
Absorbance of Control − Absorbance of Test * 100 Absorbance of Control .
International Journal of Microbiology 3

Growth Pattern.
During the growth phase, Streptomyces produces secondary metabolites, which are substances that are not strictly necessary for the organism to grow or reproduce but can provide it with a competitive edge [9]. Tese metabolites promote vegetative bacterial cells by protecting metals like iron (siderophores), protecting them from UV radiation (through coloring), thwarting competitors (antibiotics), and encouraging communication with other species [30]. Tis genetic diversity is made possible by the unusually large genome of Streptomyces, which may be triple the size of certain other bacterial genomes [31]. Past eforts have been carried out to screen and identify the potential antibioticsproducing Streptomyces species from diverse geographical locations. We have previously identifed Streptomyces species isolated from the high altitude of Nepal as a potential source of antimicrobial compounds [13]. In this study, four diferent media were used (R2YE, YEME, R5, and R5M) to grow Streptomyces sp. (G-18) and were evaluated for their antioxidant and enzyme inhibition potential. We observed the Monod growth pattern [32] with a lag phase varying from 48 to 72 hours, and Streptomyces sp. (G-18) grew exponentially until 96 hours. Te stationary phase lasted from 96 to 120 hrs, and the death phase began around 144 hrs (Figure 1). Te maximum dry weight increase was obtained on R2YE media (184 mg/L), followed by R5 media (144 mg/L), YEME media (38 mg/L), and R5M media (30 mg/L).

Antioxidant Activities.
Free radicals, especially reactive oxygen species (ROS) cause cellular damage. An abnormal buildup of free radicals is likely to arise from both endogenous and exogenous metabolism. To counteract the efects of these free radicals, antioxidants are produced in humans and other organisms [33]. Antioxidant supplementation is required to reduce high levels of ROS production and prevent harmful efects on cells, tissues, and organs that are linked to various ailments [34]. Among the analyzed extracts, our results revealed that Streptomyces sp. (G-18) grown in the R2YE medium had the highest ABTS scavenging capabilities with 82% inhibition (IC 50 � 61 ± 1.5 μg/mL), and YEME extracts showed the lowest ABTS scavenging potential with 21% inhibition (IC 50 � 370 ± 10.0 μg/mL) ( Table 1 and Figure 2). Te most DPPH radical scavenging was also observed in R2YE medium extracts with 35% inhibition (IC 50 � 240 ± 7.0 μg/mL). In contrast, the R5M medium extract did not show inhibition against DPPH (Table 1 and   4 mg/mL, respectively [36]. Based on our fndings along with previous fndings, we postulated that medium composition plays an important role in the expression of several enzymes and biomolecules that govern the activities of Streptomyces extracts.

Alpha-Amylase and Lipase Inhibition.
Of the analyzed enzymes, alpha-amylase breaks down the glycosidic linkage, releasing glucose. Hence, inhibitors of alpha-amylase help reduce glucose concentration through carbohydrate metabolism. Furthermore, obesity is linked with the abnormal breakdown and deposition of fat in the body, which is associated with several health issues such as heart disease, cancer, osteoarthritis, hypertension, and diabetes [37]. Hence, the pharmaceutical and food industries are looking for natural inhibitors. Our results with the extracts of Streptomyces sp. G-18 grown in four diferent media show potential inhibitors of enzymes, such as lipase and alphaamylase, except for the R5M medium. Among the four media, Streptomyces sp. G-18 grown in the R2YE medium revealed the highest alpha-amylase inhibition with 66% inhibition (IC 50 � 130 ± 0.5 μg/mL). However, R5M medium extract showed the least inhibition against alpha-amylase with 51% inhibition (IC 50 � 280 ± 1.0 μg/mL) ( Table 2 and Figure 4). YEME exhibited the highest lipase inhibition at 45% inhibition (IC 50 � 157 ± 6.0 μg/mL) followed by R5 and R2YE. R5M media extract revealed no inhibition of lipase (Table 2 and Figure 5). A similar work carried out by Siddharth and Vittal provided evidence of Streptomyces sp. S2A inhibits alpha-amylase and alpha-glucosidase with IC 50 values of 21 μg/mL and 20 μg/mL, respectively [38]. Statistical analysis (One-way ANOVA) revealed a signifcant diference in the results of the inhibition measurements of each extract against alpha-amylase and lipase enzymes (p < 0.05) ( Table 2). In previous studies, Cyclipostins produced by Streptomyces sp. DSM 13381 and lipstatin produced by Streptomyces toxytricini showed inhibition against lipase enzymes [39,40]. Our work is the frst to evaluate the lipase inhibitory activity of Streptomyces G-18 extracts grown in diferent media. Te extracting medium and concentration dependent inhibition pattern for enzymes indicates the importance of growth medium selection for the optimization of biochemical activities.

Tyrosinase and Elastase Inhibition.
Elastase is a protease that belongs to the chymotrypsin family and is responsible for the degradation of elastin [41], a protein that is present in the extracellular matrix (ECM). It is crucial for preventing skin aging (natural as well as premature) caused by intrinsic and extrinsic factors [42]. Te search for potent elastase inhibitors from natural sources is an alternative to commercially available chemical-based skin aging suppressors. Among the analyzed four extracts, only the R2YE medium revealed 54% elastase inhibition (IC 50 � 140 ± 7.0 μg/mL) ( Table 2). A previous study with the extract from Streptomyces KM-2753 showed noteworthy elastase inhibition [43].
Tyrosinase is a multicopper enzyme that is involved in both melanogenesis and enzymatic browning [44]. As a result, tyrosinase inhibitors may be appealing as depigmentation agents in the cosmetics and pharmaceutical sectors, as well as in food and agricultural industries as antibrowning agents. To date, numerous natural, semisynthetic, and synthetic inhibitors have been produced for this purpose using various screening approaches [44]. Our study revealed that all the analyzed crude extracts to have tyrosinase inhibitory activity. R5M medium extract had the highest tyrosinase inhibitory activity, with IC 50 values of 78 ± 2.0 μg/mL (60% inhibition) followed by YEME, R5, and R2YE medium extracts (Table 2 and Figure 6). A signifcant diference in inhibition activity of our analyzed extracts and those reported previously was observed for both elastase and tyrosinase inhibition (p < 0.05). Previous work reported several Streptomyces extracts to have tyrosinase inhibition capabilities. For example, Streptomyces hiroshimensis TI-C3 was isolated from soil and showed antityrosinase activity (498 U/mL) with improved activity (905 U/mL) when glucose and malt extract were used as the only carbon and nitrogen sources, respectively [45]. Streptomyces roseolilacinus NBRC 12815 developed two antityrosinase compounds, 12815 A (IC 50 � 9 M) and B (IC 50 � 1086 M), which were tested against mushroom and mammalian tyrosinases [46]. Tese results support a potential application of Streptomyces extracts in the cosmetic industry with our emphasis that extraction medium and processes should be taken into consideration.

Cholinesterase Inhibition Activities.
Alzheimer's disease (AD) is a neurological syndrome characterized by abnormal behavior and intellectual decline [47]. Te defciency of acetylcholine and butyrylcholine in the human brain is important for the proper functioning of neuromediators [48]. Inhibiting the AChE and BChE enzymes, which are responsible for the hydrolysis of AChE and BChE neurotransmitters, has therefore become a therapeutic option for AD [49]. Alkaloids are the greatest source of AChE and International Journal of Microbiology BChE inhibitors as therapeutic agents against AD [50]. Among our analyzed crude extracts, YEME and R5M medium extracts did not reveal any inhibitory activity toward cholinesterases (Table 2). Moreover, only the R2YE medium extract showed AChE inhibition (37 ± 0.3%; IC 50 � 137 ± 1.0 μg/mL), and only the R5 medium extract revealed 33 ± 0.9% inhibition against BChE enzyme (IC 50 � 200 ± 4.0 μg/mL). A recent study showed that Geranylphenazinediol produced by Streptomyces sp. LB173 inhibited AChE in the low micromolar range [51]. Moreover, marine-derived Streptomyces sp. UTMC 1334 revealed potent AChE inhibition [52]. Also, chromenone derivatives from marine-derived Streptomyces sp. CNQ-031 showed efective cholinesterase inhibition [53]. In our study, a signifcant diference between AChE and BChE inhibition by our extracts and reference used was observed (p < 0.05) in (Table 2). Te selectivity of Streptomyces strain in producing secondary metabolites that act against these enzymes might be the reason for medium discerning inhibition potential.

Conclusion
In conclusion, Streptomyces sp. G-18 is a potential source of enzymes inhibitors for which metabolites production and regulation are largely dependent on the growth medium and culture conditions. We observed R2YE and R5 medium as the best-suited medium for optimal growth and are associated with the highest biological activities. Our results open up the possibility to fnd other major secondary metabolites produced by Streptomyces sp. G-18 for pharmaceuticals and cosmetic industries.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Te permit for soil collection from Langtang National Park, Rasuwa, Nepal, was taken from the Department of National Park and Wildlife Conservation, Kathmandu, Nepal, with reference letter number 077/078 ECO-102.

Conflicts of Interest
Te authors declare that there are no conficts of interest.

Authors' Contributions
AGC carried out the laboratory works, generated the data, analyzed the data, prepared the manuscript draft, and revised the study. SPP carried out the laboratory works, generated the data, analyzed the data, prepared the manuscript draft, and revised the study. KKK carried out the laboratory works. AK carried out the preliminary works. AB carried out the preliminary works. BPP conceptualized the study, verifed the data, revised the manuscript, and supervised the study. AGC and SPP contributed equally to this work.